Compositions for and methods of enhancing the immune response to antigens

ABSTRACT

Compositions comprising the compound of formula are provided herein. Also provided are methods of enhancing an immune response of a subject to an antigen by administering the antigen and the composition. The enhanced immune response may be an humoral immune response, a CD4+ T cell response, a CD8+ T cell response or result in activation of antigen presenting cells. Methods of enhancing the immune response by intramuscular administration of an antigen and the composition including the compound of formula I are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/992,460, filed on Dec. 5, 2007, which is incorporated hereinby reference in its entirety.

INTRODUCTION

The fundamental purpose of a vaccine is to provide lasting immunityagainst a pathological condition. Ideally, vaccines provide functionallyactive antibodies, elicit cell-mediated immunity, and activate T- andB-lymphocytes with highly specific reactivity as well as “memory” toprovide protection against further encounters with antigen.

Adjuvants are vaccine additives which nonspecifically augment the immuneresponse. The mechanisms by which adjuvants enhance the immune systemvary widely. Adjuvants may be classified as “immunomodulatory” or“antigen delivery” systems. Immunomodulatory adjuvants prime the immunesystem by regulating the action of immune cells through alteration oflymphokine production. Antigen delivery systems, on the other hand,function to deliver the antigen to the appropriate immune cells. Inaddition, adjuvants may enhance the speed or duration of an immuneresponse, modulate antibody avidity, specificity, isotype or subclassdistribution, stimulate cell mediated immunity, promote mucosalimmunity, or enhance the immune responses in immunologically immature orsenescent individuals. Adjuvants can affect the innate, humoral orcell-mediated immune response, or a combination thereof.

SUMMARY OF INVENTION

The inventors have discovered that a synthetic glycolipid of aparticular class, when used in combination with a vaccine preparation iscapable of activating both humoral and cellular immune responses whenadministered to a subject. Accordingly, the invention providescompositions comprising compounds of formula I, wherein formula I isshown below:

where R₁ and R₂ are independently selected from —H or —OH, x is aninteger from 18 to 26 and n is an integer from 10 to 15. Compositionsincluding the compound of formula I and an antigen are also provided.

In another aspect, the invention provides methods of enhancing theimmune response in a subject to an antigen by administering acomposition including the compound of formula I and an antigen. Theenhanced immune response may be a humoral immune response, a CD4+ T cellresponse, a CD8+ cytotoxic T cell response or activation of antigenpresenting cells (APCs). The immune response of the subject is enhancedrelative to an appropriate control.

In a further aspect, the compositions of the invention are administeredintramuscularly.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a graph depicting the percentage of specific lysis ofOva-specific target cells in the blood of mice injected intravenously(IV) with PBS-96, PBS-14 or PBS-11, with or without Ova.

FIG. 2 is a graph depicting the percentage of specific lysis ofOva-specific target cells in the blood of mice injected IV with αGalCer,PBS-57, PBS-96 or PBS-14, with or without Ova.

FIG. 3 is a graph depicting the specific lysis of Ova-specific targetcells in the blood of mice injected intramuscularly (IM) with αGalCer,PBS-57, PBS-96 or PBS-14, with or without Ova.

FIG. 4A is a graph depicting accumulation of IFNγ in sera of mice 24hours after inoculation with varying concentrations of PBS-57. FIG. 4Bis a graph depicting accumulation of IFNγ in sera of mice 24 hours afterinoculation with varying concentrations of PBS-14. FIG. 4C is a graphdepicting accumulation of IFNγ in sera of mice 24 hours afterinoculation with varying concentrations of PBS-96. FIG. 4D is a graphdepicting accumulation of IFNγ in sera of mice 24 hours afterinoculation with varying concentrations of αGalCer.

FIG. 5 is a graph comparing accumulation of IFNγ in the sera of mice 24hours after inoculation with 100 ng of PBS-57, PBS-96, PBS-14 or PBS-11.

FIG. 6 is a graph depicting specific lysis of Ova-specific target cellsin the blood of mice injected IV with PBS-11, PBS-57, PBS-96 or PBS-14,with or without Ova.

FIG. 7 is a graph showing specific lysis of OVA-specific target cells inmice injected IM with 100 ng of the indicated adjuvant, with or withoutOVA.

FIG. 8 is a graph showing specific lysis of OVA-specific target cells inmice injected IM with 10 ng of the indicated adjuvant, with or withoutOVA.

FIG. 9 is a graph showing percentages of CD8+ T cells responsive to theSIINFEKL pentamer in mice injected IM with 1 μg of the indicatedadjuvant, with or without OVA.

FIG. 10 is a graph showing percentages of CD8+ T cells responsive to theSIINFEKL pentamer in mice injected IM with 100 ng of the indicatedadjuvant, with or without OVA.

FIG. 11 is a graph showing titers of IgG1 in the blood of mice injectedIM with 100 ng of the indicated adjuvant, with or without OVA.

FIG. 12 is a graph showing titers of IgG2a in the blood of mice injectedIM with 100 ng of the indicated adjuvant, with or without OVA.

FIG. 13 depicts structural formulae for PBS-14, PBS-96, PBS-11, PBS-57and αGalCer.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Adjuvants enhance the immunogenicity of antigens in vaccine preparationsin a variety of ways. An effective adjuvant also would be useful forcombination with a wide variety of antigens to enhance the immuneresponse elicited by administration of the antigen. For example, in thecase of toxins, a good humoral immune response is required. In the caseof intracellular bacteria, a cell-mediated response, mediated mainly bycytotoxic T cells and Th1 cells, is important. In the case of viralinfections, both humoral and cellular responses are fundamental tocontrol the infection. The ability of an adjuvant to enhance not onlythe humoral but also the cell-mediated immune response increases thelikelihood of developing long-lasting immunity.

Lipid species have been investigated for adjuvant properties. A numberof natural and synthetic lipid molecules are processed byantigen-presenting cells and presented by CD1 molecules to NKT cells.The prototypical compound used to study NKT cell activation in vitro andin vivo is KRN7000, an α-galactosyceramide (“αGalCer”) derived frommarine sponge Agelas mauritianus. Additional compounds recentlyidentified include isoglobotrihexosylceramide (“iGB3”) which is anendogenous glycolipid and modified 6″amino 6″ deoxygalactosyceramides,as described in PCT Application PCT/U.S.07/66250, the disclosure ofwhich is incorporated herein by reference. These compounds activate NKTcells and upregulate cytokine responses in vitro. However, in thecontext of in vivo vaccinations, little is known regarding theeffectiveness of lipid adjuvanticity for these compounds.

The inventors have found that glycosphingolipids of formula I containingan amino group and a saturated fatty acid chain, unexpectedly have theability to stimulate both a cell-mediated and humoral immune response invivo. In addition, compounds of formula I are able to stimulate animmune response against a weak nominal antigen to produce antibodies andsimultaneously provide for cell-mediated lysis of cells expressingspecific surface antigens. Two compounds of formula I, designated PBS-96and PBS-14, were shown to stimulate both cell-mediated and humoralimmune responses in vivo. These compounds also stimulated an immuneresponse against a weak nominal antigen to produce antibodies and elicita cell-mediated response. In addition, these compounds were found tostimulate a more robust response when injected intramuscularly or atlower doses as compared to other glycoshingolipids such as PBS-57 andαGalCer.

In one embodiment, the invention provides a composition comprising acompound of formula I, where formula I is:

where R₁ and R₂ are independently selected from —H or —OH, x is aninteger from 18 to 26 and n is an integer from 10 to 15. Compounds offormula I suitably have an amide group at the C6 position of thegalactose or glucose molecule and a saturated acyl chain at the ceramideportion of the compound. The composition may further include aphysiological acceptable carrier. A “physiologically acceptable” carrieris any carrier that is suitable for in vivo administration (e.g., oral,transdermal or parenteral administration) or in vitro use, i.e., cellculture. Suitable physiologically acceptable carriers for in vivoadministration include water, buffered solutions and glucose solutions,among others. Additional components of the compositions can includeexcipients such as stabilizers, preservatives, diluents, emulsifiers orlubricants in addition to the physiologically acceptable carrier.Suitable excipients include, but are not limited to, Tween 20, DMSO,sucrose, L-histadine, polysorbate 20 and serum. Suitably the compound offormula I is formulated in a liposome. Suitably the liposome is a typeSUV comprised of phosphatidyl choline (PC)/phosphatidyl glycerol(PG)/Cholesterol in a ratio of 8 μmoles/2 μmoles/5 μmoles/1 mg. Thoseskilled in the art will appreciate the compound of formula I may beformulated in a variety of ways for administration to a subject.

In another embodiment, the invention provides methods of enhancing animmune response to an antigen in a subject by administering acomposition containing the compound of formula I and the antigen. Asused herein, a “subject” is a mammal, e.g., a mouse, or more suitably ahuman. “Enhancing the immune response” refers to the ability of thecompound to enhance the humoral and/or cell mediated immune response ofa subject to the antigen in relation to a suitable control. Increasedactivation of antigen presenting cells is also included as an enhancedimmune response of the subject. For purposes of determining whether theimmune response is enhanced relative to a control, a quantitativecomparison of the signal in a sample from a subject vaccinated withantigen and the compound can be compared to the signal in a sample froma subject vaccinated with antigen alone. The immune response to theantigen may be measured in a variety of ways which will be apparent tothose skilled in the art. In the Examples, the immune response ismeasured by way of a cytotoxic specific cell lysis assay, a pentamerbinding assay, or an ELISA assay, the performance of which is routine tothose skilled in the art.

In particular embodiments, the immune response is enhanced at least 25%,at least 30%, at least 50%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, at least 150%, at least 200%, at least 400%, at least 500%,at least 750% or at least 1000%, relative to a suitable control. Asuitable control is a subject who has been administered an antigen butnot a composition of the invention. Percent enhancement may becalculated using the following formula:

[(value representing subject's immune response after treatment withcomposition containing the compound of formula I)−(value representingimmune response of control)/(value representing subject's immuneresponse after treatment with composition containing the compound offormula I)]×100.

As used herein, the terms “administration”, “co-administration” and“co-administering” refer to administration of the adjuvant and theantigen concurrently, i.e., simultaneously in time, or sequentially,i.e., administration of the adjuvant followed by administration of theantigen, or administration of the antigen followed by administration ofthe adjuvant. After administration of the adjuvant or antigen, the othercomponent can be administered substantially immediately thereafter orafter an effective time period thereafter; the effective time period isthe amount of time given for realization of maximum benefit from theadministration of the components. Alternatively, the adjuvant andantigen may be co-formulated.

The antigen may be a polypeptide, polynucleotide, or carbohydratemoiety, or combinations thereof, for example, a glycoprotein. Theantigen is suitably derived from an infectious agent (e.g., a pathogenicmicroorganism), a tumor, an endogenous molecule (e.g., a “self”molecule), or, for purposes of study, a nominal antigen, such asovalbumin (referred to herein as “OVA”). Suitably the antigen isencompassed within a vaccine. Vaccine compositions are suitablyformulated to include the compound of formula I. “Vaccine” refers to acomposition which, when administered to a subject, induces cellular orhumoral immune responses. Pharmaceutical compositions used inconjunction with the invention suitably include the compound of formulaI and a vaccine. In some embodiments, the pharmaceutical compositionsused in conjunction with the invention suitably include PBS-96 and anantigen or PBS-14 and an antigen. The structures of PBS-96 and PBS-14are shown in FIG. 13. PBS-96 and PBS-14 activate NKT cells in vitro andin vivo. Both PBS-96 and PBS-14 contain an amide group at the C6position of the galactose and a saturated acyl chain at the ceramideportion of the compound. PBS-96 and PBS-14 enhance the CD8+ T cellresponse to an antigen and PBS-96 and PBS-14 induce the release of IFN-γin vivo. In addition, PBS-96 and PBS-14 are unexpectedly superior toother glycosphingolipids when used at lower concentrations and also wheninjected intramuscularly.

Compositions including compounds of formula I may be formulated using avariety of preparative methods and inactive ingredients known to thoseof skill in the art. (Remington's Pharmaceutical Sciences, MackPublishing Co., (2000), which is incorporated herein by reference.)Compositions of the invention may also contain a suitable antigendelivery system to target the antigen to immune cells. Antigen deliverysystems are known in the art, and include, but are not limited to, MVA(Modified virus ankara), adenovirus, lentivirus, translocated subunit ofpertussis or shiga toxin, or antigen encapsulated liposomes. Effectivedosages of the compound of formula I in a vaccine composition may bedetermined by those of skill in the art, but typically range from about1 nanogram to about 10,000 micrograms per kilogram of body weight,although they are typically about 1,000 micrograms or less per kilogramof body weight. In some embodiments, the effective dosage ranges fromabout 10 nanograms to about 1,000 micrograms per kilogram of bodyweight. In another embodiment, the effective dosage ranges from about100 nanograms to about 500 micrograms per kilogram of body weight. Inanother embodiment, the effective dosage ranges from about 1 microgramto about 250 micrograms per kilogram of body weight. For purposes ofstudy, a suitable dosage for a mouse is from 1 ng to 1 μg compound offormula I per 100 μl dose depending on route of administration. Forexample, dosage of about 100 ng is suitable for intravenous injection ina mouse and dosage of as little as 10 ng was shown to be effective forintramuscular injection. The composition comprising the compound offormula I can be administered in a single dose, or split into multipledoses over a period of weeks or months.

One or more antigens may be included in the compositions or may beformulated independently. As used herein, an “antigen” refers to amolecule that stimulates an immune response in a subject to which it hasbeen administered. It will be appreciated that the dosage of antigenwill depend on the specific antigen, and on the age and immune status ofthe subject, as well as other relevant factors that may be determined bythose skilled in the art.

Whole microorganisms or portions thereof (e.g., membrane ghosts; crudemembrane preparations, lysates and other preparations of microorganisms)may be utilized as antigens. Suitably, antigens are derived fromattenuated or killed infectious agents. Suitable infectious agents fromwhich an antigen may be derived include, but are not limited to,pathogens and microorganisms such as bacteria, parasites and viruses. Insome contexts, suitable antigens are obtained or derived from a viralpathogen that is associated with human disease including, but notlimited to, HIV/AIDS (Retroviridae, e.g., gp120 molecules for HIV-1 andHIV-2 isolates, HTLV-I, HTLV-11), influenza viruses (Orthomyxoviridae,e.g., types A, B and C), herpes (e.g., herpes simplex viruses, HSV-1 andHSV-2 glycoproteins gB, gD and gH), rotavirus infections (Reoviridae),respiratory infections (parainfluenza and respiratory syncytialviruses), Poliomyelitis (Picornaviridae, e.g., polioviruses,rhinoviruses), measles and mumps (Paramyxoviridae), Rubella(Togaviridae, e.g., rubella virus), hepatitis (e.g., hepatitis virusestypes A, B, C, D, E and/or G), cytomegalovirus (e.g., gB and gH),gastroenteritis (Caliciviridae), Yellow and West Nile fever(Flaviviridae), Rabies (Rhabdoviridae), Korean hemorrhagic fever(Bunyaviridae), Venezuelan fever (Arenaviridae), warts (Papillomavirus),simian immunodeficiency virus, encephalitis virus, varicella zostervirus, Epstein-Barr virus, and other virus families, includingCoronaviridae, Birnaviridae and Filoviridae.

Suitable bacterial and parasitic antigens can also be obtained orderived from known disease-causing agents and may be used incompositions to vaccinate against diseases including, but not limitedto, diphtheria, pertussis, tetanus, tuberculosis, bacterial or fungalpneumonia, otitis media, gonorrhea, cholera, typhoid, meningitis,mononucleosis, plague, shigellosis or salmonellosis, Legionnaires'disease, Lyme disease, leprosy, malaria, hookworm, onchocerciasis,schistosomiasis, trypanosomiasis, leishmaniasis, giardiases, amoebiasis,filariasis, Borrelia, and trichinosis. Still further antigens can beobtained or derived from unconventional pathogens such as the causativeagents of kuru, Creutzfeldt-Jakob disease (CJD), scrapie, transmissiblemink encephalopathy, and chronic wasting diseases, or from proteinaceousinfectious particles such as prions that are associated with mad cowdisease.

Additional specific pathogens from which antigens can be derived includeM. tuberculosis, Chlamydia, N. gonorrhoeae, Shigella, Salmonella, Vibriocholerae, Treponema pallidum, Pseudomonas, Bordetella pertussis,Brucella, Francisella tularensis, Helicobacter pylori, Leptospirainterrogans, Legionella pneumophila, Yersinia pestis, Streptococcus(types A and B), pneumococcus, meningococcus, Haemophilus influenza(type b), Toxoplasma gondii, Moraxella catarrhalis, donovanosis, andactinomycosis; fungal pathogens include candidiasis and aspergillosis;parasitic pathogens include Taenia, flukes, roundworms, amebiasis,giardiasis, Cryptosporidium, Schistosoma, Pneumocystis carinii,trichomoniasis and trichinosis. The present invention can also be usedto provide a suitable immune response against numerous veterinarydiseases, such as foot-and-mouth diseases, coronavirus, Pasteurellamultocida, Helicobacter, Strongylus vulgaris, Actinobacilluspleuropneumonia, Bovine Viral Diarrhea Virus (BVDV), Klebsiellapneumoniae, E. coli, and Bordetella pertussis, parapertussis andbrochiseptica.

In other embodiments, antigens for inclusion in compositions that may beused in conjunction with the present invention are tumor-derivedantigens or autologous or allogeneic whole tumor cells. Suitably, thetumor antigen is a tumor specific antigen (TSA) or a tumor associatedantigen (TAA). Several tumor antigens and their expression patterns areknown in the art and can be selected based on the tumor type to betreated. Non-limiting examples of tumor antigens include cdk4(melanoma), β-catenin (melanoma), caspase-8 (squamous cell carcinoma),MAGE-1 and MAGE-3 (melanoma, breast, glioma), tyrosinase (melanoma),surface Ig idiotype (e.g., BCR) (lymphoma), Her-2/neu (breast, ovarian),MUC-1 (breast, pancreatic) and HPV E6 and E7 (cervical carcinoma).Additional suitable tumor antigens include prostate specific antigen(PSA), sialyl Tn (STn), heat shock proteins and associated tumorpeptides (e.g., gp96), ganglioside molecules (e.g., GM2, GD2, and GD3),Carcinoembryonic antigen (CEA) and MART-1.

As appreciated by skilled artisans, pharmaceutical compositions aresuitably formulated to be compatible with the intended route ofadministration. Examples of suitable routes of administration includeparenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular,oral (e.g., inhalation), enteral, transdermal (topical), transmucosal,and rectal administration. As shown in the Examples, the compounds offormula I were found to provide an unexpectedly robust enhancement ofthe immune response after intramuscular administration.

Another embodiment of the invention is a method of stimulating a humoralimmune response to an antigen. The method includes co-administering acompound of formula I and an antigen to a subject, as described above.As used herein, a “humoral immune response” refers to the production ofantibodies by B cells, and the accessory process that accompanies it,including, but not limited to, e.g., Th2 activation and cytokineproduction, germinal center formation and isotype switching, affinitymaturation production and memory cell generation. For purposes ofdetermining whether a humoral immune response is activated, aquantitative comparison of the signal in a sample from a subjectadministered antigen and a compound of formula I can be compared to asample from a subject administered antigen alone. The humoral immuneresponse may be evaluated by measuring the effector functions ofantibodies, including pathogen or toxin neutralization, classicalcomplement activation, and opsonin promotion of phagocytosis andpathogen elimination. The antibodies produced in response toco-administering the compound of formula I and an antigen may be of anytype, e.g., IgM, IgA, or IgG (such as IgG1 or IgG2). The humoral immuneresponse may be assayed by any quantitative method known in the art,e.g., ELISA, single radial immunodiffusion assay (SRID), enzymeimmunoassay (EIA), or hemagglutination inhibition assay (HAI).

A further embodiment of the invention is a method of activating CD4+ Tlymphocytes in a subject. As understood in the art, CD4+ T cells, or “Thelper cells,” are cells that recognize antigens presented by class IImajor histocompatibility marker (MHC) on the surface of antigenpresenting cells, and secrete lymphokines to stimulate bothcell-mediated and antibody-mediated branches of the immune system. CD4+T cell activation promotes lymphokine secretion, immunoglobulin isotypeswitching, affinity maturation of the antibody response, macrophageactivation and enhanced activity of natural killer (NK) and cytotoxic Tcells (CTL). Lymphokines are proteins secreted by lymphocytes thataffect their own activity and/or the activity of other cells.Lymphokines include, but are not limited to, interleukins and cytokines,e.g., IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, or IFNγ. For purposes ofdetermining whether a CD4+ T lyphocytes are activated, a quantitativecomparison of the signal in a sample from a subject vaccinated withantigen and a compound of formula I can be compared to a sample from asubject vaccinated with antigen alone. Methods to assay activation CD4+T cells are known in the art.

Another embodiment of the invention is a method of activating CD8+ Tlymphocytes in a subject. CD8+ T lymphocytes recognize antigenspresented by Class I MHC molecules (present on all nucleated cells).Engagement of the MHC class-I peptide complex results in delivery oflytic granules to the target cell causing lysis of the target cell.Methods used to assay the activation of CD8+ T cells are known in theart, and include, but are not limited to, ELISPOT, ELISA, FACS analysisfor tetramer/pentamer binding, and cytotoxic assays. Alternatively, amouse model may be used to monitor the activation of CD8+ T cells usinga fluorescent assay to measure cell-mediated cytotoxicity, as describedin Hermans et al, 2004, Journal of Immunologic Methods, 285:25-40,incorporated by reference in its entirety. In this assay, mice areimmunized on day 0 with the vaccine with or without the test compound.Syngeneic target cells are created by isolating splenocytes from asecond set of mice and labeling the cells with two separatecell-labeling fluorescent dyes or high and low concentrations of asingle fluorescent dye, e.g., CFSE or CMTMR. One set of target cells isloaded with antigen-specific peptides while a second set of target cellsis loaded with an irrelevant peptide. The two target cell populationsare mixed in equal amounts and injected into immunized mice. 24 hourslater, mice are sacrificed, and splenocytes and blood samples areobtained. The levels of each set of target cells are analyzed by flowcytometry. Activation of CD8+ lymphocytes is determined by comparing thenumber of target cells in a sample vaccinated with antigen and testcompound to the number of target cells in a sample from a subjectvaccinated with antigen alone.

Other aspects of the invention will become apparent by consideration ofthe following non-limiting examples and accompanying figures.

EXAMPLES Example 1 Testing for Enhancement of the CD8+ T Cell Responseby PBS-96, PBS-14 and PBS-11 Injected Intravenously in a Mouse Model

A mouse model was used to test in vivo specific cytotoxic T cellresponse (CD8+) elicited by test adjuvant compounds in combination withantigen when administered intravenously. Eight groups of 3 mice wereimmunized on day 0 with antigen (Ovalbumin, Ova, grade VII, Sigma, St.Louis, Mo.) with or without adjuvant, adjuvant alone, or carrier alone(control) in a total of 100 μl PBS intravenously (lateral tail vein).Test adjuvant compounds were 1 μg PBS-96, 1 μg PBS-14, 1 μg PBS-11 withor without 50 μg Ovalbumin (OVA) antigen.

Syngeneic target cells were prepared by isolating splenocytes from asecond set of C57/B1/6J CD45.2 female mice and labeling the cells witheither low concentration (0.6 μM over 8 min at 37° C.) or highconcentration (6 μM over 8 minutes at 37° C.) of CFSE (fluorescent dye).The population labeled with high concentration CFSE was pre-loaded with5 μM SIINFEKL peptide (Ova-specific peptide, NeoMPS, Inc, San Diego,Calif.) over 60 minutes at 37° C. The population labeled with lowconcentration CFSE was pre-loaded with 5 μl LCMV gp33-41 peptide(non-Ova peptide, NeoMPS, Inc, San Diego, Calif.) over 60 minutes at 37°C. Target cells were mixed with a final ratio of 47/53 of lowconcentration CFSE loaded cells to high concentration CFSE loaded cells(2×10⁷ cells total per 100 μl) and injected intravenously into each ofthe immunized mice on day 10. Mice were sacrificed at day 11, and spleencells and blood samples from the orbital sinus were collected. The meanpercentage survival of the peptide-pulsed target cells (CFSE labeledhigh concentration) were calculated relative to the control populationby flow cytometric analysis. Cytotoxic activity was expressed as percentspecific lysis (100 minus the mean percent survival of peptide-pulsedtargets). FIG. 1 depicts the percentage of specific lysis of targetcells in the blood of immunized mice. Only administration of thecombinations of Ova and PBS-96 or PBS-14 resulted in cytotoxic lysis ofOva-specific target cells. In contrast, administration of PBS-11 did notresult in specific lysis of cells.

Example 2 Comparison of Enhancement of the CD8+ T Cell Response byPBS-96, PBS-14, PBS-11, PBS-57 and αGalCer when Injected Intravenouslyand Intramuscularly in a Mouse Model

To determine the ability of the test adjuvant compounds to induce an invivo specific cytotoxic T cell response (CD8+) when administered incombination with antigen, test adjuvant compounds were further assayedusing the method described in Example 1. Eighteen groups of mice whereimmunized on day 0 either intravenously (IV) (groups 1-9 of 3 mice pergroup) or intramuscularly (groups 10-18 of 6 mice per group) as follows:

-   -   Group 1: 400 μg of Ova into 100 μl of PBS by IV;    -   Group 2: 1 μg of αGalCer into 100 μl of PBS by IV;    -   Group 3: 1 μg of PBS-57 into 100 μl of PBS by IV;    -   Group 4: 1 μg of PBS-14 into 100 μl of PBS by IV;    -   Group 5: 1 μg of PBS-96 into 100 μl of PBS by IV;    -   Group 6: 400 μg of Ova+1 μg of αGalCer into 100 μl of PBS by IV;    -   Group 7: 400 μg of Ova+1 μg of PBS-57 into 100 μl of PBS by IV;    -   Group 8: 400 μg of Ova+1 μg of PBS-14 into 100 μl of PBS by IV;    -   Group 9: 400 μg of Ova+1 μg of PBS-96 into 100 μl of PBS by IV;    -   Group 10: 400 μg of Ova into 50 μl of PBS by IM;    -   Group 11: 1 μg of αGalCer into 50 μl of PBS by IM;    -   Group 12: 1 μg of PBS-57 into 50 μl of PBS by IM;    -   Group 13: 1 μg of PBS-14 into 50 μl of PBS by IM;    -   Group 14: 1 μg of PBS-96 into 50 μl of PBS by IM;    -   Group 15: 400 μg of Ova+1 μg of αGalCer into 50 μl of PBS by IM;    -   Group 16: 400 μg of Ova+1 μg of PBS-57 into 50 μl of PBS by IM;    -   Group 17: 400 μg of Ova+1 μg of PBS-14 into 50 μl of PBS by IM;    -   Group 18: 400 μg of Ova+1 μg of PBS-96 into 50 μl of PBS by IM.

Target cells were mixed with a final ratio of 50/50 of low concentrationCFSE loaded cells to high concentration CFSE loaded cells (1×10⁷ cellseach concentration, 2×10⁷ cells total per 100 μl) and injectedintravenously into each of the immunized mice on day 10. On day 11, micewere sacrificed and blood samples were collected from the orbital sinus.Cell lysis of the OVA-specific target cells was monitored by flowcytometry of the peripheral blood cells. Specific cell lysis wasdetermined as described above.

FIG. 2 shows the results for mice injected intravenously. The averageOva-specific cytotoxic response in mice treated with Ova alone was11.8±14.4%, with Ova and αGalCer was 79.9±0.8%, in mice treated with Ovaand PBS-57 was 88.1±6.2%, in mice treated with Ova and PBS-14 was83.3±6.1%, and in mice treated with Ova and PBS96 was 89.2±10.3%.Results showed PBS-14 and PBS-96 were as effective as PBS-57 in inducingin vivo OVA-specific cytotoxic responses.

FIG. 3 shows the results for mice injected intramuscularly. The averageOVA-specific cytotoxic response in mice treated with Ova alone was1.60±14.33%, in mice treated with OVA and αGalCer was −5.85±11.01%, inmice treated with Ova and PBS-57 was 56.11±13.34%, in mice treated withOva and PBS-14 was 52.07±29.56%, and in mice treated with OVA and PBS-96was 50.29±42.6%. These results demonstrate that PBS-96 and PBS-14 bothelicit an immune response as effectively as PBS-57 both intravenouslyand intramuscularly and that PBS-14, PBS-96 and PBS-57 are moreeffective than αGalCer after intramuscular injection.

Example 3 In Vivo Stimulation of IFNγ by Test Adjuvant Compounds

To test the ability of the adjuvant test compounds to stimulate in vivocytokine release, C57BL/6 mice were administered the compounds atdifferent concentrations intravenously and the production of IFNγ insera was measured 24 hours later by ELISA. Groups of 3 mice wereinoculated intravenously (tail vein) on day 0 as follows:

-   -   Group 1: 100 μl of PBS alone    -   Group 2: 1 μg of αGalCer in 100 μl of PBS    -   Group 3: 100 ng of αGalCer in 100 μl of PBS    -   Group 4: 1 ng of αGalCer in 100 μl of PBS    -   Group 5: 0.1 ng of αGalCer in 100 μl of PBS    -   Group 6: 100 ng of αGalCer and 100 μg of Ova in 100 μl of PBS    -   Group 7: 1 μg of PBS-57 in 100 μl of PBS    -   Group 8: 100 ng of PBS-57 in 100 μl of PBS    -   Group 9: 1 ng of PBS-57 in 100 μl of PBS    -   Group 10: 0.1 ng of PBS-57 in 100 μl of PBS    -   Group 11: 100 ng of PBS-57 and 400 μg of Ova into 100 μl of PBS    -   Group 12: 1 μg of PBS-14 in 100 μl of PBS    -   Group 13: 100 ng of PBS-14 in 100 μl of PBS    -   Group 14: 1 ng of PBS-14 in 100 μl of PBS    -   Group 15: 0.1 ng of PBS-14 in 100 μl of PBS    -   Group 16: 100 ng of PBS-14 and 400 μg of Ova in 100 μl of PBS    -   Group 17: 1 μg of PBS-96 in 100 μl of PBS    -   Group 18: 100 ng of PBS-96 in 100 μl of PBS    -   Group 19: 1 ng of PBS-96 in 100 μl of PBS    -   Group 20: 0.1 ng of PBS-96 in 100 μl of PBS    -   Group 21: 100 ng of PBS-96 and 400 μg of Ova in 100 μl of PBS.        24 hours after inoculation, blood samples were collected from        the mice and IFNγ levels were detected by ELISA kit. Two ELISA        kits were used, Quantikine mouse IFNγ (RD systems) was used to        test all samples and ELISA mIFNγ (Diaclone) was used to test        group 1, 2, 3, 6, 7, 8, 11, 12, 13, 16, 17, 18 and 21. All sera        were diluted before use in ELISA as follows:

Group 1: 1/1 Group 2: 1/50 Group 3: 1/50 Group 4: 1/20 Group 5: 1/10Group 6: 1/50 Group 7: 1/50 Group 8: 1/50 Group 9: 1/20 Group 10: 1/10Group 11: 1/50 Group 12: 1/50 Group 13: 1/50 Group 814: 1/20 Group 15:1/10 Group 16: 1/50 Group 17: 1/50 Group 18: 1/50 Group 19: 1/20 Group20: 1/10 Group 21: 1/50Results are expressed as IFNγ concentration (pg/ml) in sera and takeinto account the dilution factor. FIG. 4 depicts results using theQuantikine mouse IFNγ kit by RD systems. FIG. 4A depicts results of IFNγlevels for mice immunized with PBS-57, FIG. 4B depicts results of IFNγlevels for mice immunized with PBS-14, FIG. 4C depicts results of IFNγlevels for mice immunized with PBS-96 and FIG. 4D depicts results ofIFNγ levels for mice immunized with αGalCer. At 0.1 ng, all adjuvantcandidates induce cytokine release, but mice immunized with αGalCerproduced three- or four-fold less IFNγ than mice immunized with PBS-57,PBS-14 or PBS-96 (1540.57±397.53 pg/ml, 4398.05±880.86 pg/ml,6669.31±1231.82 pg/ml, 5823.33±720.69 pg/ml respectively). At 1 ng testadjuvant compound, PBS-57, PBS-14 and PBS-96 (11425.98±833.04 pg/ml,7481.15±3454.03 pg/ml and 6271.95±3737.53 pg/ml, averaged, respectively)showed a larger response than αGalCer (average of 3802.99±586.02 pg/ml).At 100 ng of test adjuvant compound, PBS-57, PBS-96 and PBS-14 allproduced higher IFNγ levels than αGalCer (21432.76±4312.76 pg/ml forPBS-57, 19679.89±1443.48 pg/ml for PBS-96, 19582.18±3421.20 pg/ml forPBS-14, and 7714.37±3529.07 pg/ml for αGalCer, averaged). At 1 μg testcompound, PBS-57 showed a weaker response (3353.45±867.57 pg/ml)compared to the dose of 100 ng (21432.76±4312.76 pg/ml) while PBS-14 orPBS-96 still showed a lower but still robust response (16392.53±5957.70pg/ml and 17720.11±2869.97 pg/ml respectively) than at the dose of 100ng (19582.18±3421.20 pg/ml and 19679.89±1443.48 pg/ml respectively).

Another set of mice were used to compare the ability of adjuvantcompounds to stimulate in vivo cytokine release by the method describedabove. Five groups of C57BL/6 mice were administered 100 ng of PBS-57,PBS-14, PBS-96, or PBS-11 in 100 μl PBS or 100 μl PBS aloneintravenously. The production of IFNγ in sera was measured 24 hourslater by ELISA. FIG. 5 depicts results for mice immunized with 100 ng ofPBS-11, PBS-96, PBS-14 and PBS-57.

Overall, administration of PBS-14 and PBS-96 give a similar IFNγresponse to PBS-57, and an unexpectedly greater response thanadministration of PBS-11.

Example 4 Comparison of the Enhancement of the CD8+ T Cell Response byPBS-96, PBS-14, PBS-11, and PBS-57

To determine the ability of the test adjuvant compounds to induce invivo specific cytotoxic T cell response (CD8+) in combination withantigen, the test compounds were tested by the method described inExample 1. Nine groups of mice where immunized on day 0 intravenously(IV) as follows:

-   -   Group 1: 400 μg of Ova into 100 μl of PBS;    -   Group 2: 1 μg of PBS-11 into 100 μl of PBS;    -   Group 3: 1 μg of PBS-57 new formulation into 100 μl of PBS;    -   Group 4: 1 μg of PBS-14 into 100 μl of PBS;    -   Group 5: 1 μg of PBS-96 into 100 μl of PBS;    -   Group 6: 400 μg of Ova+1 μg of PBS-11 into 100 μl of PBS;    -   Group 7: 400 μg of Ova+1 μg of PBS-57 new formulation into 100        μl of PBS;    -   Group 8: 400 μg of Ova+1 μg of PBS-14 into 100 μl of PBS;    -   Group 9: 400 μg of Ova+1 μg of PBS-96 into 100 μl of PBS.        Target cells were mixed with a final ratio of 50/50 of low        concentration CFSE loaded cells to high concentration CFSE        loaded cells (1×10⁷ cells each concentration, 2×10⁷ cells total        per 100 μl) and injected intravenously into each of the        immunized mice on day 10. On day 11, mice were sacrificed and        blood samples were collected from the orbital sinus. Specific        cell lysis of the Ova-specific target cells was monitored by        flow cytometry of the peripheral blood cells. Specific cell        lysis was determined as described above. Results are shown in        FIG. 6. The average Ova-specific cell lysis was 11.8±14.4% for        mice treated with Ova alone, 32.3±2.5% for mice treated with Ova        and PBS-11, 88.1±6.2% in mice treated with Ova and PBS-57,        83.3±6.1% for mice treated with Ova and PBS-14, and 89.2±10.3%        in mice treated with Ova and PBS-96. These results demonstrate        that PBS-14 and PBS-96 are as effective as PBS-57 at inducing an        in vivo cytotoxic response after intravenous administration in        combination with antigen.

Example 5 Comparison of the Enhancement of the CD8+ T Cell Response byDecreasing Amounts of Adjuvant After Intramuscular Injection

To determine the relative ability of the test adjuvant compounds toenhance the immune response, a similar experiment to that described inExample 2 was performed. In this experiment mice were injectedintravenously with decreasing amounts of adjuvant (100 ng and 10 ng,respectively) in combination with 50 μg of OVA antigen at day 0 asfollows:

Experiment A:

-   -   Group 1: 50 μg of Ova into 100 μl PBS    -   Group 2: 100 ng αGalCer;    -   Group 3: 100 ng PBS-1    -   Group 4: 100 ng PBS-14    -   Group 5: 100 ng PBS-57    -   Group 6: 100 ng PBS-96    -   Group 7: 50 μg of Ova with 100 ng αGalCer;    -   Group 8: 50 μg of Ova with 100 ng PBS-11    -   Group 9: 50 μg of Ova with 100 ng PBS-14    -   Group 10: 50 μg of Ova with 100 ng PBS-57    -   Group 11: 50 μg of Ova with 100 ng PBS-96

Experiment B:

-   -   Group 1: 50 μg of Ova into 100 μl PBS    -   Group 2: 10 ng αGalCer;    -   Group 3: 10 ng PBS-11    -   Group 4: 10 ng PBS-14    -   Group 5: 10 ng PBS-57    -   Group 6: 10 ng PBS-96    -   Group 7: 50 μg of Ova with 10 ng αGalCer;    -   Group 8: 50 μg of Ova with 10 ng PBS-11    -   Group 9: 50 μg of Ova with 10 ng PBS-14    -   Group 10: 50 μg of Ova with 10 ng PBS-57    -   Group 11: 50 μg of Ova with 10 ng PBS-96        Target cells were administered on day 10 of the experiment and        blood was collected on day 11. The results for Experiment A        using 100 ng of each adjuvant are shown in FIG. 7 and the        results for Experiment B are shown in FIG. 8. FIGS. 7 and 8        demonstrate that PBS-14 and PBS-96 are unexpectedly better than        other adjuvants at enhancing the CD8+ T cell response to an        antigen at low doses when administered intramuscularly. In fact        after administration of OVA and only 10 ng of either PBS-14 or        PBS-96 intramuscularly the percentage of specific lysis of the        target cells by CD8+ T cells is still over 60%, while the        percentage of specific lysis of target cells after        administration of OVA and the same amount of PBS-57, PBS-11 or        αGalCer was indistinguishable from the controls.

Example 6 Comparison of the Enhancement of the CD8+ T Cell Response byDecreasing Amounts of Adjuvant After Intramuscular Injection

To verify the results obtained using the in vivo cytotoxicity assaydescribed in the Examples above, similar experiments were performed andCD8+ T cell activation was determined by measuring the percentage ofOVA-specific CD8+ T cells using a pentamer assay. Briefly, mice wereinjected intramuscularly with the indicated amounts of OVA and testadjuvant compound at either 1 μg or 100 ng per mouse, respectively onday 0 as follows:

Experiment A:

-   -   Group 1: 100 μl of PBS;    -   Group 2: 50 μg of Ova into 100 μl PBS;    -   Group 3: 50 μg of Ova with 1 μg αGalCer;    -   Group 4: 50 μg of Ova with 1 μg PBS-11    -   Group 5: 50 μg of Ova with 1 μg PBS-14    -   Group 6: 50 μg of Ova with 1 μg PBS-57    -   Group 7: 50 μg of Ova with 1 μg PBS-96

Experiment B:

-   -   Group 1: 100 μl of PBS;    -   Group 2: 50 μg of Ova into 100 μl PBS;    -   Group 3: 50 μg of Ova with 100 ng αGalCer;    -   Group 4: 50 μg of Ova with 100 ng PBS-11    -   Group 5: 50 μg of Ova with 100 ng PBS-14    -   Group 6: 50 μg of Ova with 100 ng PBS-57    -   Group 7: 50 μg of Ova with 100 ng PBS-96        A second injection was administered to the mice on day 14 and        blood was collected from the mice on day 21. The lymphocytes        were collected and analyzed by FACS analysis using H-2 K^(b)        SIINFEKL pentamer and CD8 antibody to detect CD8+ T cells        responsive to OVA. The results of Experiment A are shown in FIG.        9 and the results of Experiment B are shown in FIG. 10. FIG. 9        demonstrates that when administered at 1 μg PBS-14, PBS-96 and        PBS-57 enhanced the percentage of OVA specific CD8+ T cells        after vaccination, while PBS-11 and αGalCer were not as        effective. FIG. 10 demonstrates that at the lower dose of 100 ng        PBS-14 and PBS-96 were surprisingly much better at enhancing the        CD8+ T cell response to an antigen as compared to PBS-57, PBS-11        and αGalCer.

Example 7 Comparison of the Enhancement of the Humoral ResponseFollowing Intramuscular Immunization with Adjuvant and Antigen

To evaluate whether the humoral and CD4+ T helper cell immune responseswere also enhanced by administration of the test adjuvants with antigen,the IgG1 and IgG2a antibody responses were measured in mice vaccinatedwith OVA with or without the test adjuvants. Mice (6 per group) wereinjected intramuscularly with 50 μg of OVA either alone or incombination with 100 ng of the indicated adjuvants (Freund's adjuvantwas used as a positive control) as follows:

-   -   Group 1: 500 μg of Ova with CFA/IFA (Positive control)    -   Group 2: 50 μg of Ova    -   Group 3: 50 μg of Ova and 100 ng of αGalCer    -   Group 4: 50 μg of Ova and 100 ng of PBS-11    -   Group 5: 50 μg of Ova and 100 ng of PBS-14    -   Group 6: 50 μg of Ova and 100 ng of PBS-57    -   Group 7: 50 μg of Ova and 100 ng of PBS-96        At 14 days post-injection, blood samples were collected and        ELISAs for IgG1 and IgG2a were performed using mouse monoclonal        antibodies specific for OVA isotype IgG1 or IgG2a. The results        are shown as the titer of the antibody in peripheral blood in        ng/ml. The results for IgG1 are depicted in FIG. 11 and those        for IgG2a are depicted in FIG. 12. As shown in FIG. 11, PBS-14,        PBS-96 and PBS-57 were all able to elicit a robust IgG1 titer        and enhanced the OVA-specific IgG1 titer as compared to        vaccination with OVA alone or OVA in combination with PBS-11 or        αGalCer. Surprisingly, PBS-14 and PBS-96 enhanced the OVA        specific IgG2a titer about as well as Freund's adjuvant and much        better than PBS-57.

While the compositions and methods of this invention have been describedin terms of exemplary embodiments, it will be apparent to those skilledin the art that variations may be applied to the compositions andmethods and in the steps or in the sequence of steps of the methodsdescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention. In addition, allpatents and publications listed or described herein are incorporated intheir entirety by reference.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to acomposition containing “a polynucleotide” includes a mixture of two ormore polynucleotides. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise. All publications, patents and patentapplications referenced in this specification are indicative of thelevel of ordinary skill in the art to which this invention pertains. Allpublications, patents and patent applications are herein expresslyincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference. In case of conflict between the presentdisclosure and the incorporated patents, publications and references,the present disclosure should control.

It also is specifically understood that any numerical value recitedherein includes all values from the lower value to the upper value,i.e., all possible combinations of numerical values between the lowestvalue and the highest value enumerated are to be considered to beexpressly stated in this application.

1. A composition comprising a compound of formula I:

wherein R₁ is H or —OH, R₂ is —H or —OH, x is an integer from 18 to 26,and n is an integer from 10 to
 15. 2. The composition of claim 1,wherein x is
 23. 3. The composition of claim 2, wherein n is
 13. 4. Thecomposition of claim 1, wherein x is
 21. 5. The composition of claim 4,wherein n is
 13. 6. The composition of claim 1, further comprising anantigen.
 7. A pharmaceutical composition comprising the composition ofclaim 1 and an antigen.
 8. A method of enhancing an immune response of asubject to an antigen comprising administering the antigen and thecomposition of claim 1 to the subject, wherein the immune response ofthe subject to the antigen is enhanced relative to an immune response ofa control to the antigen.
 9. The method of claim 8, wherein the immuneresponse of the subject is enhanced at least 50% relative to thecontrol.
 10. The method of claim 8, wherein the immune response of thesubject is enhanced at least 100% relative to the control.
 11. Thenmethod of claim 8, wherein the immune response of the subject isenhanced at least 100% relative to the control.
 12. The method of claim8, wherein the antigen and the composition of any claim 1 areadministered concurrently.
 13. The method of claim 8, wherein thecomposition is administered via a route selected from the groupconsisting of intravenously, intramuscularly, subcutaneously,intradermally, intraperitoneally, intranasally and by inhalation.
 14. Amethod of enhancing a humoral immune response of a subject to an antigencomprising administering the antigen and the composition of claim 1 tothe subject, wherein the humoral immune response of the subject to theantigen is enhanced relative to an immune response of a control to theantigen.
 15. The method of claim 14, wherein the humoral immune responsecomprises production of IgG antibodies.
 16. The method of claim 14,wherein the humoral immune response comprises production of IgAantibodies.
 17. A method of enhancing a CD4+ T cell response of asubject to an antigen comprising administering the antigen and thecomposition of claim 1 to the subject, wherein the immune response ofthe subject to the antigen is enhanced relative to a CD4+ T cellresponse of a control to the antigen.
 18. The method of claim 17,wherein the enhanced CD4+ T cell response comprises activation of CD4+ Tlymphocytes.
 19. The method of claim 18, wherein activation of the CD4+T lymphocytes comprises an increase in a Th1 immune response.
 20. Themethod of claim 18, wherein activation of the CD4+ T lymphocytescomprises an increase in a Th2 immune response.
 21. The method of claim18, wherein activation of the CD4+ T lymphocytes comprises an increasein both a Th1 and a Th2 immune response.
 22. A method of enhancing aCD8+ T cell response of a subject to an antigen comprising administeringthe antigen and the composition of claim 1 to the subject, wherein theimmune response of the subject to the antigen is enhanced relative to aCD8+ T cell response of a control to the antigen.
 23. The method ofclaim 22, wherein the enhanced CD8+ T cell response comprises activationof the CD8+ T lymphocytes.
 24. The method of claim 23, wherein theactivation of the CD8+ T lymphocytes comprises an increase in cytotoxicresponse.
 25. A method of enhancing activation of antigen presentingcells of a subject to an antigen comprising administering the antigenand the composition of claim 1 to the subject, wherein the immuneresponse of the subject to the antigen is enhanced relative toactivation of antigen presenting cells of a control to the antigen. 26.A method of enhancing an immune response of a subject to an antigencomprising administering the antigen and the composition of claim 1 tothe subject intramuscularly, wherein the immune response of the subjectto the antigen is enhanced relative to an immune response of a controlto the antigen.
 27. The method of claim 26, wherein the immune responseof the subject is enhanced at least 50% relative to the control.
 28. Themethod of claim 26, wherein the immune response of the subject isenhanced at least 100% relative to the control.
 29. Then method of claim26, wherein the immune response of the subject is enhanced at least1000% relative to the control.
 30. The method of claim 26, wherein theantigen and the composition are administered concurrently.